Biomedical subjects

Jyoti J Watters

Publications and source records attributed to Jyoti J Watters.

12 recordsLinked to original sources

PubMed2006

Purinergic receptors modulate MAP kinases and transcription factors that control microglial inflammatory gene expression.

Following many types of brain injury, microglial cell hyperactivation, and the subsequent release of neurotoxic mediators into the CNS contributes to inflammation and neuronal death. Among the proteins important for modulating the inflammatory function of microglia are the P2 purinergic receptors for which extracellular adenine nucleotides, such as ATP, are ligands. Because adenine nucleotides are abundant in the extracellular fluid following brain injury, ATP may represent an important component of the inflammatory microenvironment controlling microglial cell function. Although much work has been done examining the mechanisms whereby adenine nucleotides stimulate inflammatory mediator production, little is known concerning their complementary inhibitory effects. In this review we will focus on what is currently known about the microglial inhibitory effects of adenine nucleotides in the context of inflammation and summarize the current knowledge of their effects via purinergic receptors on microglial signal transduction pathways including transcription factors important for controlling inflammatory gene expression. The relevance of these mechanisms to microglial inflammatory function and physiology will be discussed. Further, we present data here illustrating that MAP kinase signal transduction pathways are altered in activated microglia that have been primed with or co-exposed to adenine nucleotides; effects that are stimulus- and MAPK pathway-specific. We also demonstrate the ability of P2X7 receptors to stimulate the phosphorylation of CREB, a putative inhibitory transcription factor in microglia. Together, these data indicate that ATP may be an endogenous inhibitor or neuroprotective molecule decreasing the inflammatory capacity of microglia.

Adenosine Triphosphate↗

PubMed2005

Minocycline exerts inhibitory effects on multiple mitogen-activated protein kinases and IkappaBalpha degradation in a stimulus-specific manner in microglia.

CNS inflammation mediated by microglial activation can result in neuronal and glial cell death in a variety of neurodegenerative and demyelinating diseases. Minocycline, a second-generation tetracycline, has profound anti-inflammatory properties in the CNS mediated, in part, by inhibition of microglia. MAPK and nuclear factor-kappaB (NF-kappaB) activation are hallmarks of activated microglia and they are critical for the expression of many inflammatory mediators. In the present study, we investigated minocycline effects on activation of p38, c-Jun-N-terminal activated protein kinase (JNK) 1/2 and extracellular signal regulated kinase (ERK) 1/2 MAPKs and inhibitor alpha of NF-kappaB (IkappaBalpha) degradation in BV-2 and primary microglial cells. Our results demonstrate that minocycline has the ability to inhibit all MAPKs but these effects strongly depend on the stimulus used for MAPK activation. Minocycline significantly decreased activation of all lipopolysaccharide-stimulated MAPKs but it was without effect on MAPKs activated by H2O2. Minocycline inhibited JNK1/2 and ERK1/2 but not p38 when stimulated by 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate, indicating that minocycline affects only certain upstream signaling target(s) that are stimulus-specific. Our data also suggest that protein kinase C (PKC) inhibition may be partially involved in the minocycline mechanism of MAPK inhibition. In addition, minocycline attenuated lipopolysaccharide-stimulated degradation of IkappaBalpha implying a possible inhibitory role on NF-kappaB transcriptional activity.

Adenosine Triphosphate↗

PubMed2005

Microglia function in brain tumors.

Microglia play an important role in inflammatory diseases of the central nervous system (CNS). These cells have also been identified in brain neoplasms; however, as of yet their function largely remains unclear. More recent studies designed to characterize further tumor-associated microglia suggest that the immune effector function of these cells may be suppressed in CNS tumors. Furthermore, microglia and macrophages can secrete various cytokines and growth factors that may contribute to the successful immune evasion, growth, and invasion of brain neoplasms. A better understanding of microglia and macrophage function is essential for the development of immune-based treatment strategies against malignant brain tumors.

Animals↗

PubMed2005

Prolactin and estrogen enhance the activity of activating protein 1 in breast cancer cells: role of extracellularly regulated kinase 1/2-mediated signals to c-fos.

Despite the important roles of both prolactin (PRL) and 17beta-estradiol (E2) in normal mammary development as well as in breast cancer, and coexpression of the estrogen receptor (ER) and PRL receptor in many mammary tumors, the interactions between PRL and E2 in breast cancer have not been well studied. The activating protein 1 (AP-1) transcription factor, a known regulator of processes essential for normal growth and development as well as carcinogenesis, is a potential site for cross-talk between these hormones in breast cancer cells. Here we demonstrate that PRL and E2 cooperatively enhance the activity of AP-1 in MCF-7-derived cells. In addition to the acute PRL-induced ERK1/2 activation, PRL and E2 also individually elicited delayed, sustained rises in levels of phosphorylated p38 and especially ERK1/2. Together, these hormones increased the dynamic phosphorylation of ERK1/2 and c-Fos, and induced c-fos promoter activity. Synergistic activation of the transcription factor, Elk-1, reflected the PRL-E2 interaction at ERK1/2 and is a likely mechanism for activation of the c-fos promoter via the serum response element. The enhanced AP-1 activity resulting from the interaction of these hormones may increase expression of many target genes that are critical for oncogenesis and may contribute to neoplastic progression.

Breast Neoplasms↗

PubMed2005

Purinergic receptor modulation of BV-2 microglial cell activity: potential involvement of p38 MAP kinase and CREB.

ATP is abundant in the extracellular fluid following brain injury, and it exerts potent modulatory effects on microglia, whose hyperactivation is thought to exacerbate neuronal damage. We show here that ATP decreases LPS-stimulated iNOS and COX-2 expression and reduces NO release in BV-2 microglia by a mechanism involving p38 MAP kinase. Further, we demonstrate that the inhibitory effects of ATP on NO production occur within 30 min of exposure and correlate with activation of the transcription factor CREB. Together, these data suggest that ATP may exert neuroprotective effects in the brain via a mechanism involving augmented activation of the p38/CREB pathway.

Adenine Nucleotides↗

PubMed2005

Cervical spinal cord injury upregulates ventral spinal 5-HT2A receptors.

Following chronic C2 spinal hemisection (C2HS), crossed spinal pathways to phrenic motoneurons exhibit a slow, spontaneous increase in efficacy by a serotonin (5-HT)-dependent mechanism associated with 5-HT2A receptor activation. Further, the spontaneous appearance of cross-phrenic activity following C2HS is accelerated and enhanced by exposure to chronic intermittent hypoxia (CIH). We hypothesized that chronic C2HS would increase 5-HT and 5-HT2A receptor expression in ventral cervical spinal segments containing phrenic motoneurons. In addition, we hypothesized that CIH exposure would further increase 5-HT and 5-HT2A receptor density in this region. Control, sham-operated, and C2HS Sprague-Dawley rats were studied following normoxia or CIH (11% O2-air; 5-min intervals; nights 7-14 post-surgery). At 2 weeks post-surgery, ventral spinal gray matter extending from C4 and C5 was isolated ipsilateral and contralateral to C2HS. Neither C2HS nor CIH altered 5-HT concentration measured with an ELISA on either side of the spinal cord. However, 5-HT2A receptor expression assessed with immunoblots increased in ipsilateral gray matter following C2HS, an effect independent of CIH. Immunocytochemistry revealed increased 5-HT2A receptor expression on identified phrenic motoneurons (p<0.05), as well as in the surrounding gray matter. Contralateral to injury, 5-HT2A receptor expression was elevated in CIH, but not normoxic C2HS rats (p<0.05). Our data are consistent with the hypothesis that spontaneous increase in 5-HT2A receptor expression on or near phrenic motoneurons contributes to strengthened crossed-spinal synaptic pathways to phrenic motoneurons following C2HS.

Animals↗

PubMed2004

Multiple kinase cascades mediate prolactin signals to activating protein-1 in breast cancer cells.

The importance of prolactin (PRL) in physiological proliferation and differentiation of the mammary gland, together with high levels of PRL receptors in breast tumors, the association of circulating PRL with incidence of breast cancer, and the recognition of locally produced PRL, point to the need for greater understanding of PRL actions in mammary disease. Although PRL has been shown to activate multiple kinase cascades in various target cells, relatively little is known of its signaling pathways in the mammary gland apart from the Janus kinase 2/ signal transducer and activator of transcription 5 pathway, particularly in tumor cells. Another potential effector is activating protein-1 (AP-1), a transcription complex that regulates processes essential for neoplastic progression, including proliferation, survival and invasion. We demonstrate that PRL activates AP-1 in MCF-7 cells, detectable at 4 h and sustained for at least 24 h. Although Janus kinase 2 and ERK1/2 are the primary mediators of PRL-induced signals, c-Src, phosphatidylinositol 3'-kinase, protein kinase C, and other MAPKs contribute to maximal activity. PRL activation of these pathways leads to increased c-Jun protein and phosphorylation, JunB protein, and phosphorylation of c-Fos, elevating the levels of AP-1 complexes able to bind DNA. These active AP-1 dimers may direct expression of multiple target genes, mediating some of PRL's actions in mammary disease.

Breast Neoplasms↗

PubMed2004

Estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor beta.

Estrogens are well known to exert antiinflammatory effects outside the central nervous system (CNS). They have also been shown to exert neuroprotective effects in the CNS after several types of injury, including neurodegeneration. However, the molecular mechanisms by which these effects occur remain unclear. Because microglial hyperactivation and their production of neurotoxins is associated with many types of brain injury for which estrogens are beneficial, we sought to investigate the ability of estrogen to modulate microglial function. Furthermore, because little is known regarding the role of each of the two known estrogen receptors (ERs) in microglia, our studies were designed to test the hypothesis that 17beta-estradiol (E(2)) exerts antiinflammatory effects in microglia, specifically via interactions with ERbeta. We tested this hypothesis using the murine microglial cell line BV-2, which naturally expresses only ERbeta. Our results indicate that not only does E(2) decrease lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, it also reduces the expression of cyclooxygenase-2, a target for estrogen that has not previously been reported for ERbeta. We also observed that LPS-stimulated TNFalpha mRNA was increased by estrogen. E(2) exerts these effects within 30 min compared with typical estrogen transcriptional responses. Tamoxifen and ICI 182,780 differentially blocked the inhibitory effects of E(2) on LPS-stimulated iNOS and cyclooxygenase-2. In addition, we show that E(2) alters LPS-stimulated MAPK pathway activation, supporting the idea that alterations in the MAPKs may be a potential mechanism by which ERbeta mediates decreased microglial activation.

Animals↗

PubMed2004

The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho.

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

Actin Cytoskeleton↗

PubMed2003

Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages.

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X(7). In this regard, after exposure to bacterial LPS, P2X(7) activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X(7) has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-kappa B, we tested the hypothesis that LPS and nucleotides regulate NF-kappa B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X(7)-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-kappa B DNA-binding activity and a sustained attenuation of levels of the NF-kappa B inhibitory protein I kappa B alpha. Interestingly, a persistent reduction in I kappa B alpha levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating I kappa B alpha protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in I kappa B alpha levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-kappa B/I kappa B-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-kappa B and Ras/MEK/MAPK-associated pathways.

Adenosine Triphosphate↗

PubMed2003

BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia.

Intermittent hypoxia causes a form of serotonin-dependent synaptic plasticity in the spinal cord known as phrenic long-term facilitation (pLTF). Here we show that increased synthesis of brain-derived neurotrophic factor (BDNF) in the spinal cord is necessary and sufficient for pLTF in adult rats. We found that intermittent hypoxia elicited serotonin-dependent increases in BDNF synthesis in ventral spinal segments containing the phrenic nucleus, and the magnitude of these BDNF increases correlated with pLTF magnitude. We used RNA interference (RNAi) to interfere with BDNF expression, and tyrosine kinase receptor inhibition to block BDNF signaling. These disruptions blocked pLTF, whereas intrathecal injection of BDNF elicited an effect similar to pLTF. Our findings demonstrate new roles and regulatory mechanisms for BDNF in the spinal cord and suggest new therapeutic strategies for treating breathing disorders such as respiratory insufficiency after spinal injury. These experiments also illustrate the potential use of RNAi to investigate functional consequences of gene expression in the mammalian nervous system in vivo.

Animals↗

PubMed2002

A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production.

Endotoxin (lipopolysaccharide, LPS) is a component of the outer membrane of Gram-negative bacteria and promotes the activation of macrophages and microglia. Although these cells are highly LPS-responsive, they serve unique tissue-specific functions and exhibit different LPS sensitivities. Accordingly, it was of interest to evaluate whether these biological differences reside in variations within LPS signaling pathways between these two cell types. Because the mitogen-activated protein kinases ERK-1 and ERK-2 have been implicated in the control of many immune responses, we tested the concept that they are a key indicator for differences in cellular LPS sensitivity. We observed that murine RAW 264.7 macrophages and murine BV-2 microglial cells both respond to LPS by exhibiting increased IkappaBalpha degradation, enhanced NF-kappaB DNA binding activity, and elevated nitric oxide and interleukin-1beta production. Although LPS potently stimulates ERK activation in RAW 264.7 macrophages, it does not activate ERK-1/-2 in BV-2 microglia. Moreover, antagonism of the MEK/ERK pathway potentiates LPS-stimulated nitric oxide production, suggesting that LPS-stimulated ERK activation can exert inhibitory effects in macrophage-like cells. These data support the idea that ERK activation is not a required function of LPS-mediated signaling events and illustrate that alternative/additional pathways for LPS action exist in these cell types.

Animals↗